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US8012487B2 - Synthetic peptides and DNA sequences for treatment of multiple sclerosis - Google Patents

Synthetic peptides and DNA sequences for treatment of multiple sclerosis Download PDF

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US8012487B2
US8012487B2 US10/492,794 US49279402A US8012487B2 US 8012487 B2 US8012487 B2 US 8012487B2 US 49279402 A US49279402 A US 49279402A US 8012487 B2 US8012487 B2 US 8012487B2
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mog
gene
mbp
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Avraham Ben-Nun
Nicole Kerlero De Rosbo
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Yeda Research and Development Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to synthetic peptides and polypeptides and synthetic human genes useful for the treatment of multiple sclerosis as well as for diagnostic purposes.
  • Autoimmune diseases result from the immune system's failure to maintain self-tolerance to antigen(s) in the affected organ. Over 40 systemic and organ-specific autoimmune diseases have been observed. Among the organ-specific autoimmune diseases are multiple sclerosis, myasthenia gravis, thyroiditis, insulin-dependent diabetes mellitus, rheumatoid arthritis and others. In spite of major and significant advances in molecular and cellular immunology in the last two decades, the molecular basis for self-tolerance and the mechanisms regulating it are still a major challenge in immunology, and autoimmune diseases remain a major medical problem. The immune-specific approaches to therapy of the disease, expected to be the most effective, have not yet yielded an effective therapy for any of the autoimmune diseases.
  • the major difficulty in devising immune specific approaches to therapy lies in the complexity of the autoimmune diseases, particularly with regard to the multiplicity of target antigens and because of the possibility that the primary target antigen(s) may be different in different patients, the difficulty in determining which of the possible target antigens is the primary target antigen for each patient, and against which of the possible epitopes on that protein the pathogenic autoimmune response is primarily directed. This is further complicated by the likely “spread of autoimmunity” as disease develops.
  • MS multiple sclerosis
  • CNS central nervous system
  • EAE experimental autoimmune encephalomyelitis
  • myelin basic protein MBP
  • proteolipid protein PGP
  • myelin oligodendrocyte glycoprotein MOG
  • Activated CD4+ T cells specific for MBP or PLP are sufficient to cause EAE upon their transfer into naive syngeneic recipients, and potentially pathogenic T cells reactive against MBP or PLP have been demonstrated in MS (reviewed in Tuohy, 1994); however, comparable T cell responses to MBP or PLP were also detected in healthy individuals (reviewed in Tuohy, 1994).
  • specific responses to these myelin antigens are likely to be of importance in the course of the disease, they may not represent the primordial pathogenic response in MS.
  • a primary target antigen in MS is CNS myelin-specific.
  • Myelin proteins such as MOG, myelin-oligodendrocytic basic protein (MOBP) and oligodendrocyte-specific protein (OSP) are believed to be specific components of CNS myelin (Gardinier et al., 1992; Yamamoto et al., 1994; Bronstein et al., 1997).
  • OSP may also be a potential target antigen for autoimmune demyelinating diseases such as MS.
  • a potential primary target antigen in MS could be defined as a CNS antigen which has an encephalitogenic potential, i.e. can cause EAE, and against which autoimmune reactivity can be detected in MS patients.
  • MBP, PLP, MOG and now also MOBP can be considered potential primary target antigens, as autoreactivity against one of these antigens may play an important role in the initiation/progression of MS.
  • the potential role of autoimmune responses to OSP in the pathogenesis of MS should also be considered.
  • autoimmune responses to other nonencephalitogenic CNS components myelin-specific or non myelin-specific, which can be detected in MS, are more likely to represent secondary events resulting from “autoimmune spread” as a result of inflammation within CNS with ongoing disease.
  • the multiplicity of potential primary target antigens in MS points to the complexity of the disease with regard to possible pathogenic processes involved, possible etiology of the disease, and most importantly, it imposes major difficulties in devising immune-specific therapeutic approaches to MS.
  • the major problems that must be addressed by immune-specific therapies for a given autoimmune disease include the multiplicity of potential primary target antigens with the possibility that the primary target antigens differ in different patients, and the recently acknowledged “spreading of autoimmunity” as disease develops. This phenomenon is described as the observation of variation in the active immunogenic epitopes with the progression of the disease. This results in the evolution of the primary T cell response focused on a particular self-antigen, towards the recruitment of T cells to multiple antigenic determinants on this or other potential target autoantigens within the affected organ (Tuohy et al., 1998; Kumar, 1998).
  • EEC immunogenic epitope clusters
  • said at least two IECs may be derived from a sole autoantigen or from at least two different autoantigens related to said autoimmune disease, and polypeptides encoded thereby, that can be used for the treatment and diagnosis of autoimmune diseases such as multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA) and others.
  • MS multiple sclerosis
  • IDDM insulin-dependent diabetes mellitus
  • RA rheumatoid arthritis
  • each gene comprising sequences coding for at least two IECs of autoantigen(s) related to a specific autoimmune disease such as MS, IDDM or RA, said synthetic gene being selected from:(i) a synthetic human target autoantigen gene (designated shTAG) comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to said autoimmune disease; and (ii) a synthetic human multitarget autoantigen gene (designated shMultiTAG) comprising nucleotide sequences coding for at least one IEC of at least two different autoantigens related to said autoimmune disease.
  • shTAG synthetic human target autoantigen gene
  • shMultiTAG synthetic human multitarget autoantigen gene
  • WO 01/31037 further disclosed several synthetic polypeptides, each polypeptide comprising amino acid sequences of at least two IECs of autoantigens related to a specific autoimmune disease such as MS, IDDM or RA, said synthetic polypeptide being selected from: (i) a synthetic human polypeptide (designated shPEP) comprising amino acid sequences of at least two IECs of a sole autoantigen related to said autoimmune disease; and (ii) a synthetic human multitarget polypeptide (designated shMultiPEP) comprising amino acid sequences of at least one IEC of at least two different autoantigens related to said autoimmune disease.
  • a synthetic human polypeptide designated shPEP
  • shMultiPEP synthetic human multitarget polypeptide
  • MS the multiplicity of potentially pathogenic autoreactivities against the myelin components, MBP, PLP, MOG, MOBP, and OSP, which have been detected in different patients, suggest that the primary target antigen(s) and/or the major epitope(s) against which the dominant pathogenic autoreactivities are directed, may differ in different patients. Because neoreactivities are also likely to emerge, disease progression may be associated with multiple potentially pathogenic T cell autoreactivities. This imposes major difficulties for devising immune-specific approaches for therapy of MS.
  • immune-specific therapy would have to be tailored for each patient according to the antigenic and epitope specificities of the potentially pathogenic autoimmune T-cells detected in that patient, including novel T-cell specificities elicited as a result of “autoimmune spread” as disease progresses.
  • An alternative and more generally applicable approach would ideally be, if all or most of relevant potentially pathogenic autoreactivities could be targeted concomitantly. This would allow immunomodulation of MS, irrespective of the antigenic primacy or dominance of the pathogenic autoimmune response in individual patients.
  • MBPAc1-11 which suppresses MBP-induced EAE in these F 1 mice, had no effect on MOG-induced EAE and a marginal therapeutic effect on EAE induced by the MBP/MOG combination (Leadbetter et al., 1998), an observation most likely related to its specific suppressive effect on MBP-reactive T cells.
  • Highly relevant to treatment of disease with multiple autoreactivities is the strong therapeutic effect on EAE of MP4, a chimeric fusion protein, comprising the whole long isoform of MBP (21.5 kDa MBP) and the hydrophilic domains of PLP ( ⁇ PLP) (Elliott et al., 1996).
  • the present invention provides a synthetic peptide comprising amino acid sequences of at least one IEC of at least one human autoantigen related to MS, said synthetic peptide being selected from:
  • an unaltered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N- and C-termini, the resulting IEC being capable of stimulating human T cells, wherein the peptides are those of SEQ ID Nos: 10 and 22-47; and
  • an altered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N-and C-termini, in which sequence 1 to 3 T-cell receptor (TCR) contact amino acid residues are substituted by a suitable amino acid such as Ala, the resulting IEC altered in the TCR residue being capable of immunomodulating the potentially pathogenic T-cell response against the epitope without risk of exacerbation, excluding altered peptides derived from the MBP 83-99 and PLP 139-151 sequences.
  • TCR T-cell receptor
  • the human autoantigen related to MS is preferably MOG, MBP, OSP, MOBP, PLP, and MAG.
  • Examples of unaltered peptides according to the invention are the MOG peptides of SEQ ID NOs: 22-25 ( FIG. 1 ), the MBP peptides of SEQ ID NOs: 26-30 ( FIG. 2 ), the OSP peptides of SEQ ID NOs: 10, 31-35 ( FIG. 3 ), the MOBP peptides of SEQ ID NOs: 36-39 ( FIG. 4 ), and PLP peptides of SEQ ID NOs: 40-47 ( FIG. 5 ).
  • Examples of altered peptides according to the invention are the MOG-AL peptides of SEQ ID NOs: 48-53 ( FIG. 15 ), the MBP-AL peptides of SEQ ID NOs: 54-61 ( FIG. 16 ), the OSP-AL peptides of SEQ ID NOs: 62-69 ( FIG. 17 ), the MOBP-AL peptides of SEQ ID NOs: 70-73 ( FIG. 18 ), and the PLP-AL peptides of SEQ ID NOs: 74-86 ( FIG. 19 ).
  • the present invention provides a synthetic gene comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS or coding for at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said IECs have amino acid sequences selected from the sequences of the unaltered and/or altered peptides of the invention, particularly those of SEQ ID NOs: 10 and 22-86.
  • the present invention polypeptides that are the protein products of the synthetic genes of the invention and comprise amino acid sequences of at least two IECs of a sole autoantigen related to MS or at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said IECs have amino acid sequences selected from the sequences of the unaltered and/or altered peptides of the invention, particularly those of SEQ ID NOs: 10 and 22-86.
  • the present invention provides pharmaceutical compositions comprising a mixture of at least two of the unaltered or altered peptides of the invention, or at least one polypeptide or a synthetic gene of the invention for treatment or diagnostic of MS.
  • FIG. 1 depicts a scheme of the herein designated shMOG gene in which the sequences of epitope clusters a-d are SEQ ID NO:22-25, respectively.
  • FIG. 2 depicts a scheme of the herein designated shMBP gene in which the sequences of epitope clusters a-e are SEQ ID NO:26-30, respectively.
  • FIG. 3 depicts a scheme of the herein designated shOSP gene in which the sequences of epitope clusters a-f are SEQ ID NO:32, 31, 33, 16, 34 and 35, respectively.
  • FIG. 4 depicts a scheme of the herein designated shMOBP gene in which the sequences of epitope clusters a-d are SEQ ID NO:36, 37, 39 and 38, respectively.
  • FIG. 5 depicts a scheme of the herein designated shPLP gene in which the sequences of epitope clusters a-h are SEQ ID NO:40-45, 47 and 46, respectively.
  • FIG. 6 depicts a scheme of the herein designated Y-MSPe gene.
  • FIG. 7 depicts a scheme of the herein designated ⁇ Y-MSPe gene.
  • FIG. 8 shows that MOG altered peptide ligands (APLs) substituted at crucial T-cell receptor (TCR) contact residues 41 and 44 suppress EAE induced with peptide MOG 37-52.
  • APLs MOG altered peptide ligands substituted at crucial T-cell receptor (TCR) contact residues 41 and 44 suppress EAE induced with peptide MOG 37-52.
  • I and M indicate incidence of disease and mortality in the group, respectively.
  • FIGS. 9A-9B show that inhibition of EAE associated with multiple pathogenic autoreactivities requires multi-epitope-directed immunomodulation.
  • 9 A Multiantigen/epitope reactivity of Y-MSPb-reactive line T cells.
  • FIG. 10 shows the MOG peptides of SEQ ID NOs: 1-4 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of MOG.
  • Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.
  • FIG. 11 shows the MBP peptides of SEQ ID NOs: 5-7 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of MBP.
  • Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.
  • FIG. 12 shows the OSP peptides of SEQ ID NOs: 8-13 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of OSP.
  • Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.
  • FIG. 13 shows the MOBP peptides of SEQ ID NOs: 14-16 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of MOBP.
  • Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.
  • FIG. 14 shows the PLP peptides of SEQ ID NOs: 17-21 with the location of the nonameric core sequences predicted to bind and fit into the MS-associated HLA-DR (broken line below the sequence) and HLA-DQ (full line above the sequence) molecules, within the selected MS-related epitope clusters of PLP.
  • Bolded and underlined amino acids represent the TCR contact residues selected to be substituted.
  • FIG. 15 depicts a scheme of the herein designated shMOG-AL gene in which the sequences of epitope clusters A-F are SEQ ID NO:48-53, respectively.
  • FIG. 16 depicts a scheme of the herein designated shMBP-AL gene in which the sequences of epitope clusters A-H are SEQ ID NO:54-61, respectively.
  • FIG. 17 depicts a scheme of the herein designated shOSP-AL gene in which the sequences of epitope clusters A-G are SEQ ID NO:62-69, respectively.
  • FIG. 18 depicts a scheme of the herein designated shMOBP-AL gene in which the sequences of epitope clusters A-D are SEQ ID NO:70-73, respectively.
  • FIG. 19 depicts a scheme of the herein designated shPLP-AL gene in which the sequences of epitope clusters A, A 1 and B-L are SEQ ID NO:74-86, respectively.
  • FIG. 20 depicts a scheme of the herein designated Y-MSP-AL gene.
  • FIG. 21 depicts a scheme of the herein designated ⁇ Y-MSP-AL gene.
  • Autoantigen refers to the self-molecules (proteins) recognized as potential target antigens in an autoimmune disease.
  • Epipe refers to an antigenic determinant of the autoantigen.
  • IEC's immunological epitopic clusters
  • the cluster may include one or more flanking, overlapping epitopes or such epitopes in tandem with one another.
  • Immunogenic (epitopic) cluster coding region is used to refer to the nucleotide sequence that encodes for an IEC.
  • Immunogenic is used herein to refer to the ability of an epitope to initiate an immune response.
  • Immunomodulatory is used to refer to the ability of an IEC to modulate, regulate, control or antagonize an autoantigenic induction of an immune response in an appropriate animal model.
  • the selection of the IECs of the identified potential target autoantigens related to MS is based on experimental identification of the epitopes most frequently recognized in patients, as assayed by reactivity to overlapping peptides of the relevant autoantigen, and/or on determination of the preferred binding mode of regions of the molecule to HLA associated with MS as predicted by computer modeling, preferably confirmed by binding assays and/or experimental data obtained in HLA-transgenic mice.
  • Table 1 summarizes IECs which have been experimentally identified or predicted to be potential epitopes for MS, as described in WO 01/31037:
  • MOG 34-56 [SEQ ID NO:1]; 67-114 [SEQ ID NO:2]; 3-27 [SEQ ID NO:3]; 205-215 [SEQ ID NO:4].
  • MBP 84-111 [SEQ ID NO:5]; 141-168 [SEQ ID NO:6]; 12-42 [SEQ ID NO:7].
  • OSP 42-73 [SEQ ID NO:8]; 98-109 [SEQ ID NO:9]; 187-206[SEQ ID NO:10]; 192-206 [SEQ ID NO:11]; 20-33 [SEQ ID NO:12]; 129-145 [SEQ ID NO:13].
  • MOBP 15-33 [SEQ ID NO:14]; 55-90 [SEQ ID NO:15]; 156-172 [SEQ ID NO:16].
  • PLP 103-150 [SEQ ID NO:17]; 177-203 [SEQ ID NO:18]; 218-240 [SEQ ID NO:19]; 38-52 [SEQ ID NO:20]; 264-276 [SEQ ID NO:21].
  • MOG 37-58 [SEQ ID NO:22]; 65-95 [SEQ ID NO:23]; 7-32 [SEQ ID NO:24]; 202-218 [SEQ ID NO:25].
  • MBP 82-103 [SEQ ID NO:26]; 136-156 [SEQ ID NO:27]; 148-170 [SEQ ID NO:28]; 7-29 [SEQ ID NO:29]; 25-45 [SEQ ID NO:30].
  • OSP 38-64 [SEQ ID NO:31]; 48-77 [SEQ ID NO:32]; 94-112 [SEQ ID NO:33]; 187-206 [SEQ ID NO:10]; 17-38 [SEQ ID NO:34]; 124-150 [SEQ ID NO:35].
  • MOBP 13-38 [SEQ ID NO:36]; 54-80 [SEQ ID NO:37]; 72-89 [SEQ ID NO:38]; 156-174 [SEQ ID NO:39].
  • APLs altered peptide ligands
  • TCR recognition is conferred by only some residues of the peptide presented by MHC class II molecules (Sette et al., 1987; Gautam et al., 1992).
  • peptide analogs of specific antigens have been generated in a variety of antigen systems by introducing single amino acid substitutions, and tested for their capacity to stimulate activation events in the T cells specific for the relevant antigenic peptide. Data resulting from such analysis have demonstrated that some of the interactions with the TCR are more critical than others.
  • the “primary TCR contact site” is defined as the amino acid focused on most intently by all T cells in a population specific for the same antigen, and this amino acid appeared to be the most critical residue of the determinant to the TCR binding. Secondary residues are defined as the other TCR contact sites, which are unique to each TCR in the population, and are assumed to play a lesser role in the overall interaction between TCR and ligand (Evavold et al., 1994; Sloan-Lancaster and Allen, 1996).
  • APLs altered peptide ligands
  • APLs ability of APLs to induce different signaling events has been shown to result in several possible processes of peripheral T cell modulation, anergy and/or induction of Th2-type cytokine secretion and/or antagonistic inhibition of proliferation to the stimulatory peptide and/or inhibition of Th1-type cytokine secretion.
  • Such attributes make APLs very attractive for the design of potential therapeutic approaches to autoimmune diseases.
  • APL altered peptide ligand
  • the human MBP 83-99 region (ENPVVHFFKNIVTPRTP) (comprised within SEQ ID NO: 26) is one of the major immunodominant MBP epitopes recognized in the context of DR2, the MHC class II haplotype most associated with MS.
  • the TCR contact residues for this epitope have been defined with F-89 apparently representing a primary TCR contact residue, while H-88 and K-91 apparently serve as secondary TCR contact residues (Wucherpfenning et al., 1994; Windhagen et al., 1995; Singh et al., 1999; Kozovska et al., 1998).
  • the predicted nonameric core sequences in each epitope cluster of each of the potential primary target antigens MOG, MBP, OSP, MOBP, and PLP are comprised within the peptides of SEQ ID NOs: 1-21, and are shown in FIGS. 10-14 (broken line below the sequence for the MS-associated HLA-DR, and full line above the sequence for the MS-associated HLA-DQ).
  • the nonameric core sequences which fit into the MS-relevant HLA-DR/DQ molecule and are flanked by 2-5 amino acids at their N- and C-termini were considered to represent potential epitopes associated with MS.
  • the peptides encompassing these predicted epitopes for each target autoantigen MOG, MBP, OSP, MOBP, and PLP are represented by SEQ ID NOs: 22-33, 10 and 34-47.
  • the TCR contact residues were predicted according to the predicted core nonameric sequence binding to the MS-relevant HLA-DR/DQ molecules.
  • the peptides encompassing predicted epitopes were modified in their TCR contact residues by replacement of one or more of said TCR contact residues by alanine (Ala), but substitutions by other suitable amino acids is also encompassed by the invention.
  • TCR contact residues were selected from the overlapping potential epitopes within the clusters, so that autoreactivity against as many overlapping epitopes would be antagonized without interfering with their MHC binding residues or turning some epitopes to super-agonists.
  • Examples of altered peptides of the invention are represented by the SEQ ID NOs: 48-86, that correspond to the peptides of SEQ ID NOs: 22-33, 10 and 34-47, respectively, but in which one or more TCR contact amino acid residues have been replaced by Ala.
  • MOG-AL peptides A-F (SEQ ID NOs: 48-53, respectively) in FIG. 15 and SEQ ID NOs: 232-233;
  • MBP-AL peptides A-H (SEQ ID NOs: 54-61, respectively) in FIG. 16 ;
  • OSP-AL peptides A-E, E′, F,G (SEQ ID NOs: 62-69, respectively)in FIG. 17 ;
  • MOBP-AL peptides A-D (SEQ ID NOs: 70-73, respectively) in FIG. 18 ;
  • PLP-AL peptides A, A′,B-L (SEQ ID NOs: 74-86, respectively) in FIG. 19 .
  • Epitopes appropriately altered in their TCR contact residues, when appropriately administered, are expected to immunomodulate the potentially pathogenic T-cell response against the epitope with minimal risk of exacerbating the pathogenic T-cell response.
  • the structure of the DQ molecule (DQA1*0102/DQB1*0602) is not known, and was modeled according to related structures (PDB codesliak, 2seb), using the MSI modeling software package (MSI Inc., San-Diego, Calif.), in particular the Homology and Discover modules.
  • MSI Inc. San-Diego, Calif.
  • a 9 ⁇ 20 binding preference matrix was also constructed for this molecule.
  • the binding matrices were used in a computer program in which a 9 amino-acid moving window is applied to the sequence of each epitope cluster of each autoantigen and a binding score is calculated for each 9 amino-acid sequence. This score is estimated as the sum of the binding preferences from the appropriate 9 ⁇ 20 matrix.
  • non MHC-binding residues preferably at position 5 and/or 2 of the predicted nonameric sequence with preferred binding mode to HLA-DR or -DQ were substituted with Ala.
  • substitution of these residues with other amino acids is also envisaged.
  • FIGS. 10-14 show the nonameric sequences within the selected epitope clusters of each autoantigen, with binding score above average which were also found to fit into the MHC binding site of HLA-DR structure or of HLA-DQ models.
  • Nonameric sequences indicated by broken lines (below sequence) were taken as representing HLA-DR binding epitopes; nonameric sequences indicated by full lines (above sequence) were taken as representing HLA-DQ binding epitopes.
  • some regions within a selected epitope cluster may contain overlapping HLA-DR and/or HLA-DQ binding epitopes.
  • alteration of one potential TCR contact residue for one epitope may not antagonize the autoimmune T-cell reactivity against another overlapping epitope. Alternatively, it may interfere with the MHC binding of another overlapping epitope, thus preventing the possibility to antagonize the potential pathogenic T-cell reactivity against this epitope(s).
  • alteration of a TCR contact residue of one epitope may affect the overlapping epitope, turning it into a super-agonist rather than an antagonist.
  • TCR contact residues may need to be altered, necessitating replicate sequences with substitutions at different positions (for example, see SEQ ID NOs: 48-86).
  • SEQ ID NOs: 48-86 The bolded and underlined amino acids in each epitope cluster shown in FIGS. 10-14 , are the TCR contact residues determined to be substituted according to the above criteria.
  • the present invention thus provides a synthetic peptide comprising amino acid sequences of at least one IEC of at least one human autoantigen related to MS, said synthetic peptide being selected from:
  • an unaltered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N- and C-termini, the resulting IEC being capable of stimulating human T cells, wherein the peptides are those of SEQ ID NOs: 10 and 22-47; and
  • an altered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N-and C-termini, in which sequence one to three T-cell receptor (TCR) contact amino acid residues are substituted by a suitable amino acid such as Ala, the resulting IEC altered in the TCR residue being capable of immunomodulating the potentially pathogenic T-cell response against the epitope without risk of exacerbation, excluding altered peptides derived from the MBP 83-99 and PLP 139-151 sequences.
  • TCR T-cell receptor
  • Altered peptides derived from the MBP 83-99 sequence are disclosed for example in U.S. Pat. No. 6,251,396 (altered MBP 83-99), U.S. Pat. No. 6,329,499 (altered MBP 86-99) and U.S. Pat. No. 6,369,033 (altered MBP 87-99).
  • Altered PLP 139-151 peptides are disclosed by Hafler, 1996 and Young, 2002.
  • the IEC is derived from at least one human autoantigen related to MS such as MOG, MBP, OSP, MOBP, PLP, and MAG.
  • Examples of altered synthetic peptides of the invention include: (i) a peptide containing MOG epitope(s) in which one to three TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 48-53; (ii) a peptide containing MBP epitope(s) in which 1-3 TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 54-61; (iii) a peptide containing OSP epitope(s) in which 1-4 TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 62-69; (iv) a peptide containing MOBP epitope(s) in which 1-3 TCR contact residues are substituted by Ala such as the peptides of SEQ ID NOs: 70-73; and (v) a peptide containing PLP epitope(s) in which 1-3 TCR contact residues are substituted
  • the present invention further provides a synthetic gene comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS or coding for at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said IECs have amino acid sequences selected from the sequences of the unaltered and/or altered peptides of the invention, for example the peptides of SEQ ID NOs: 10 and 22-86.
  • the synthetic genes comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS are herein in the specification and claims identified by a designation including the letters “sh” (standing for “synthetic human”) followed by the abbreviation of the autoantigen: shMOG, shMP, shOSP, shMOBP, and shPLP genes ( FIGS. 1-5 ).
  • shMOG the abbreviation for “altered ligand”
  • shMOG-AL the abbreviation for “altered ligand”
  • the synthetic gene comprises nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS, wherein said IECs have amino acid sequences selected from the sequences of the unaltered peptides of the invention of SEQ ID NOs: 10 and 22-47.
  • IECs have amino acid sequences selected from the sequences of the unaltered peptides of the invention of SEQ ID NOs: 10 and 22-47.
  • shMOG SEQ ID NO:102
  • shMBP SEQ ID NO:114
  • shOSP SEQ ID NO:128)
  • shMOBP SEQ ID NO:138
  • shPLP shPLP
  • the synthetic gene comprises nucleotide sequences coding for at least two IECs of a sole autoantigen related to MS, wherein said IECs have amino acid sequences selected from the sequences of the altered peptides of the invention, for example of SEQ ID NOs: 48-86.
  • IECs have amino acid sequences selected from the sequences of the altered peptides of the invention, for example of SEQ ID NOs: 48-86.
  • shMOG-AL SEQ ID NO:87
  • shMBP-AL SEQ ID NO:88
  • shOSP-AL SEQ ID NO:89
  • shMOBP-AL SEQ ID NO:90
  • shPLP-AL SEQ ID NO:91
  • amino acid sequences representing epitope clusters with altered TCR contact residues were aligned with or without addition of a linker comprised of glycine and serine (GS).
  • the synthetic gene is a multitarget autoantigen gene comprising nucleotide sequences coding for at least one IEC of at least two, preferably 3 or 4, more preferably 5 or more different autoantigens related to MS, and analogs thereof, wherein said IECs have amino acid sequences selected from the sequences of the unaltered and/or altered peptides of the invention.
  • the synthetic multitarget autoantigen genes comprising nucleotide sequences coding for at least one IEC of at least two different autoantigens related to MS, are herein in the specification and claims identified by a designation including an initial capital Y followed by the abbreviation MS for multiple sclerosis, the letter P and either a small letter “e”, when the IECs have amino acid sequences of the unaltered peptides of the invention, for example of SEQ ID NOs: 10 and 22-47, or the letters “AL”, when the IECs have amino acid sequences of the altered peptides of the invention, for example of SEQ ID NOs: 48-86.
  • the synthetic multitarget autoantigen genes preferably comprise a high number of preferred IECs of the selected autoantigens, preferably of MOG, MBP, OSP, MOBP and PLP.
  • Examples of multitarget autoantigen genes wherein the IECs have amino acid sequences of the unaltered peptides of the invention of SEQ ID NOs: 10 and 22-47 are the genes herein designated Y-MSPe (SEQ ID NO: 225) and ⁇ Y-MSPe (SEQ ID NO: 227), represented by the constructs of FIGS. 6 and 7 , respectively.
  • Examples of multitarget autoantigen genes wherein the IECs have amino acid sequences of the altered peptides of the invention of SEQ ID NOs: 48-86 are the genes herein designated Y-MSP-AL (SEQ ID NO: 92) and ⁇ Y-MSP-AL (SEQ ID NO: 93), represented by the constructs of FIGS. 20 and 21 , respectively.
  • a synthetic gene comprising nucleotide sequences coding for at least two, preferably all the six MOG amino acid sequences of the SEQ ID NOs: 48-53, each of them carrying 1-3 alanine substitutions, more preferably the shMOG-AL gene of SEQ ID NO: 87 depicted by the construct of FIG. 15 ;
  • a synthetic gene comprising nucleotide sequences coding for at least two, preferably all the eight MBP amino acid sequences of the SEQ ID NOs: 54-60, each of them carrying 1-3 alanine substitutions, more preferably the shMBP-AL gene of SEQ ID NO: 88 depicted by the construct of FIG. 16 ;
  • a synthetic gene comprising nucleotide sequences coding for at least two, preferably all the eight OSP amino acid sequences of the SEQ ID NOs: 61-69, each of them carrying 1-3 alanine substitutions, more preferably the shOSP-AL gene of SEQ ID NO: 89 depicted by the construct of FIG. 17 ;
  • a synthetic gene comprising nucleotide sequences coding for at least two, preferably all the four of the MOBP amino acid sequences of the SEQ ID NOs: 70-73, each of them carrying 1-3 alanine substitutions, more preferably the shMOBP-AL gene of SEQ ID NO: 90 depicted by the construct of FIG. 18 ;
  • a synthetic gene comprising nucleotide sequences coding for at least two, preferably all the 13 of the PLP amino acid sequences of the SEQ ID NOs: 74-86, each of them carrying 1-3 alanine substitutions, more preferably the shPLP-AL gene of SEQ ID NO: 91 depicted by the construct of FIG. 19 ;
  • a synthetic multitarget autoantigen gene comprising the nucleotide sequences of at least two of the synthetic genes of (i)-(v) above, preferably comprising all said sequences, more preferably the Y-MSP-AL gene of SEQ ID NO: 92 depicted by the construct of FIG. 20 ;
  • a synthetic multitarget autoantigen gene being the truncated form of (vi), comprising the nucleotide sequences of at least two synthetic genes selected from the group consisting of: (a) a gene comprising nucleotide sequences coding for the MOG-AL amino acid sequences of the SEQ ID NOs: 48-51; (b) a gene comprising nucleotide sequences coding for the MBP-AL amino acid sequences of the SEQ ID NOs: 54-59; (c) a gene comprising nucleotide sequences coding for the OSP-AL amino acid sequences of the SEQ ID NOs: 62-67; (d) a gene comprising nucleotide sequences coding for the MOBP-AL amino acid sequences of the SEQ ID NOs: 70-73; and (e) a gene comprising nucleotide sequences coding for the PLP-AL amino acid sequences of the SEQ ID NOs: 74-84; preferably the ⁇ Y-MSP
  • the skilled person may prepare the synthetic genes encoding the IECs of the appropriate autoantigens by standard recombinant techniques.
  • the synthetic genes may contain the coding sequences for the IECs in any particular order, for example, the coding regions for all the clusters from each autoantigen may be grouped together or, alternatively, IECs from different autoantigens may be organized randomly along the synthetic genes.
  • the former arrangement prevails and each cluster coding region may be separated from the next by 3, 6, 9 etc. nucleotides or possibly by a restriction site as shown in FIGS. 1-7 and FIGS. 15-21 .
  • the synthetic gene codes for at least one, preferably 2-3, immunogenic epitopic clusters specific for each of at least two autoantigens, though it may be more preferable to include 4 or 5 or possibly more clusters from each autoantigen.
  • the amino acid sequences representing epitope clusters with altered TCR contact residues were aligned with or without addition of a linker comprised of glycine and serine (GS).
  • Synthetic genes coding for the IECs of autoantigens related to MS are obtained stepwise.
  • a synthetic gene for each autoantigen encoding the selected IECs arranged tandemly is prepared by PCR overlap extension using overlapping synthetic oligonucleotides encoding the IECs. Briefly, 60-70 nucleotide long oligonucleotides representing codons of the amino acid residues of the selected IECs are synthesized. The oligonucleotides overlap at their 5′ and/or 3′ ends by 18 nucleotides which are complementary to their neighboring oligonucleotides.
  • Specific restriction endonuclease sites are included in the first and the last oligonucleotide to facilitate cloning, as well as to enable in frame ligation to neighboring synthetic genes (see below for the construction of the Y-MSPe and Y-MSP-AL genes).
  • DNA sequence corresponding to the aligned, linked clusters was modified where necessary, to include alterations not resulting in amino acid changes to neutralize possibly problematic restriction endonuclease sites (RES), to minimize formation of DNA secondary structures or to “bacterize” codons, as well as alterations leading to substitution of cysteine for serine in order to increase the solubility of the expressed protein.
  • DNA sequences corresponding to RES were added to enable cloning and expression, as well as to allow in frame ligation to neighbouring mini-genes.
  • a template for the synthetic gene is generated by mixing the relevant oligonucleotides (each 75 pmol) in Taq DNA polymerase buffer (40 ⁇ l final volume) containing dNTPs (RO181; MBI Fermentas AB, Vilnius, Lithuania) at a final concentration of 0.2 mM each and a mixture of 0.2 U Vent DNA polymerase and 0.2 U Taq DNA polymerase (AB-0192; Advanced Biotechnologies, Surrey, UK). After denaturation (94° C., 1 min) and annealing of the oligonucleotides through their complementary ends (55° C., 2 min), PCR overlap extension is carried out at 72° C.
  • the resulting template (4 ⁇ l) is amplified by PCR at standard conditions for 30 cycles, using the relevant oligonucleotides as 5′ and 3′ reverse primers.
  • the amplified PCR product of the expected size is eluted from agarose gel and directly cloned into a T vector (pGEM-T, A3600; Promega Corp., Madison, Wis., USA).
  • the constructed synthetic gene is cleaved out from the pGEM-T/synthetic gene plasmid and subcloned into the bacterial expression vector pRSET (V351-20; Invitrogen, San Diego, Calif., USA) via NheI and BglII, 3′ to its 6xHis tag, using standard molecular biology techniques. DNA sequence analysis is performed using the pRSET-specific primers to confirm the synthetic gene DNA sequence as an open reading frame with the ATG of the pRSET expression vector.
  • the shMOG DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 102 and SEQ ID NO: 103, respectively.
  • the shMBP gene ( FIG. 2 ) was constructed using for template generation the following primers: MBP.p1 (SEQ ID NO: 104), MBP.p2(rev), (SEQ ID NO: 105), MBP.p3 (SEQ ID NO: 106), MBP.p4(rev) (SEQ ID NO: 107), MBP.p5 (SEQ ID NO: 108), MBP.p6(rev) (SEQ ID NO: 109), MBP.p7 (SEQ ID NO: 110), MBP.p8(rev) (SEQ ID NO: 111), and for amplification the 5′ and 3′ reverse primers MBP.p1a (SEQ ID NO: 112), and MBP.p8a(rev) (SEQ ID NO: 113).
  • the shMBP DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 114 and SEQ ID NO: 115, respectively.
  • the shOSP gene ( FIG. 3 ) was constructed using for template generation the following primers: OSP.p1 (SEQ ID NO: 116), OSP.p2 (rev) (SEQ ID NO: 117), OSP.p3 (SEQ ID NO: 118), OSP.p4 (rev) (SEQ ID NO: 119), OSP.p5 (SEQ ID NO: 120), OSP.p6 (rev) (SEQ ID NO: 121), OSP.p7 (SEQ ID NO: 122), OSP.p8 (rev) (SEQ ID NO: 123), OSP.p9 (SEQ ID NO: 124), OSP.p10 (rev) (SEQ ID NO: 125), and for amplification the 5′ and 3′ reverse primers OSP.p1a (SEQ ID NO: 126), and OSP.p10a (rev) (SEQ ID NO: 127).
  • the shOSP DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 128
  • the shMOBP gene ( FIG. 4 ) was constructed using for template generation the following primers: MOBP.p1 (SEQ ID NO: 130), MOBP.p2 (rev) (SEQ ID NO: 131), MOBP.p3 (SEQ ID NO: 132), MOBP.p4 (rev) (SEQ ID NO: 133), MOBP.p5 (SEQ ID NO: 134), MOBP.p6 (rev) (SEQ ID NO: 135), and for amplification the 5′ and 3′ reverse primers MOBP.p1a (SEQ ID NO: 136), and MOBP.p6a (rev) (SEQ ID NO: 137).
  • the shMOBP DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 138 and SEQ ID NO: 139, respectively.
  • the shPLP gene ( FIG. 5 ) was constructed using for template generation the following primers: PLP.p1 (SEQ ID NO: 140), PLP.p2 (rev) (SEQ ID NO: 141), PLP.p3 (SEQ ID NO: 142), PLP.p4 (rev) (SEQ ID NO: 143), PLP.p5 (SEQ ID NO: 144), PLP.p6 (rev) (SEQ ID NO: 145), PLP.p7 (SEQ ID NO: 146), PLP.p8 (rev) (SEQ ID NO: 147), PLP.p9 (SEQ ID NO: 148), PLP.p10 (rev) (SEQ ID NO: 149), PLP.p11 (SEQ ID NO: 150), PLP.p12 (rev) (SEQ ID NO: 151), and for amplification the 5′ and 3′ reverse primers PLP.p1a (SEQ ID NO: 152), and PLP.p12a (rev) (S
  • the shMOG-AL gene ( FIG. 15 ) was constructed using for template generation the following primers: MOG.AL.p1 (SEQ ID NO: 156), MOG.AL.p2 (rev) (SEQ ID NO: 157), MOG.AL.p3 (SEQ ID NO: 158), MOG.AL.p4 (rev) (SEQ ID NO: 159), MOG.AL.p5 (SEQ ID NO: 160), MOG.AL.p6 (rev) (SEQ ID NO: 161), MOG.AL.p7 (SEQ ID NO: 162), MOG.AL.p8 (rev) (SEQ ID NO: 163), and for amplification the 5′ and 3′ reverse primers MOG.AL.p1a (SEQ ID NO: 164), and MOG.AL.p8a (rev) (SEQ ID NO: 165).
  • the shMOG-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 87 and SEQ ID NO: 166
  • the shMBP-AL gene ( FIG. 16 ) was constructed using for template generation the following primers: MBP.AL.p1 (SEQ ID NO: 167), MBP.AL.p2 (rev) (SEQ ID NO: 168), MBP.AL.p3 (SEQ ID NO: 169), MBP.AL.p4 (rev) (SEQ ID NO: 170), MBP.AL.p5 (SEQ ID NO: 171), MBP.AL.p6 (rev) (SEQ ID NO: 172), MBP.AL.p7 (SEQ ID NO: 173), MBP.AL.p8 (rev) (SEQ ID NO: 174), MBP.AL.p9 (SEQ ID NO: 175), MBP.AL.p10 (rev) (SEQ ID NO: 176), and for amplification the 5′ and 3′ reverse primers MBP.AL.p1a (SEQ ID NO: 177), and MBP.AL.p10a (rev) (SEQ ID NO:
  • the shOSP-AL gene ( FIG. 17 ) was constructed using for template generation the following primers: OSP.AL.p1 (SEQ ID NO: 180), OSP.AL.p2 (rev) (SEQ ID NO: 181), OSP.AL.p3 (SEQ ID NO: 182), OSP.AL.p4 (rev) (SEQ ID NO: 183), OSP.AL.p5 (SEQ ID NO: 184), OSP.AL.p6 (rev) (SEQ ID NO: 185), OSP.AL.p7 (SEQ ID NO: 186), OSP.AL.p8 (rev) (SEQ ID NO: 187), OSP.AL.p9 (SEQ ID NO: 188), OSP.AL.p10 (rev) (SEQ ID NO: 189), OSP.AL.p11 (SEQ ID NO: 190), OSP.AL.p12 (rev) (SEQ ID NO: 191), and for amplification the 5′ and 3′ reverse primers O
  • the shMOBP-AL gene ( FIG. 18 ) was constructed using for template generation the following primers: MOBP.AL.p1 (SEQ ID NO: 195), MOBP.AL.p2 (rev) (SEQ ID NO: 196), MOBP.AL.p3 (SEQ ID NO: 197), MOBP.AL.p4 (rev) (SEQ ID NO: 198), MOBP.AL.p5 (SEQ ID NO: 199), MOBP.AL.p6 (rev) (SEQ ID NO: 200), and for amplification the 5′ and 3′ reverse primers MOBP.AL.p1a (SEQ ID NO: 201), and MOBP.AL.p6a (rev) (SEQ ID NO: 202).
  • the shMOBP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 90 and SEQ ID NO: 203, respectively.
  • the shPLP-AL gene ( FIG. 19 ) was constructed using for template generation the following primers: PLP.AL.p1 (SEQ ID NO: 204), PLP.AL.p2 (rev) (SEQ ID NO: 205), PLP.AL.p3 (SEQ ID NO: 206), PLP.AL.p4 (rev) (SEQ ID NO: 207), PLP.AL.p5 (SEQ ID NO: 208), PLP.AL.p6 (rev) (SEQ ID NO: 209), PLP.AL.p7 (SEQ ID NO: 210), PLP.AL.p8 (rev) (SEQ ID NO: 211), LP.AL.p9 (SEQ ID NO: 212), PLP.AL.p10 (rev) (SEQ ID NO: 213), PLP.AL.p11 (SEQ ID NO: 214), PLP.AL.p12 (rev) (SEQ ID NO: 215), PLP.AL.p13 (SEQ ID NO:
  • a synthetic multitarget autoantigen gene of the invention encoding randomly organized EECs of several autoantigens is prepared by ligating together the resulting synthetic genes for each autoantigen in one open reading frame to form the desired multiple gene.
  • the shMOG, shMBP, shOSP, shMOBP and shPLP genes of Examples 1-5 above are ligated sequentially via specific endonuclease restriction sites which have been incorporated to allow their ligation in one open reading frame as shown in the construct depicted in FIG. 6 .
  • the pGEM-T/shMOG is cleaved at the BamHI and HindIII sites, the shMBP gene is excised from pGEM-T/shMBP with BamHI and SpeI, the shOSP gene is excised from pGEM-T/shOSP with SpeI and PstI, the shMOBP gene is excised from pGEM-T/shMOBP with PstI and XhoI, and the shPLP gene is excised from pGEM-T/shPLP with XhoI and HindIII.
  • the DNA fragments of the right sizes are eluted from agarose gel, cleaned and sequential ligations are carried out to link the shMBP gene to the shMOG gene via their BamHI sites, the shOSP gene to the shMBP gene via their SpeI sites, the shMOBP gene to the shOSP gene via their PstI sites, and the shPLP gene to the shMOBP gene via their XhoI sites and to the pGEM-T/shMOG via their HindIII sites, as depicted in FIG. 6 .
  • the resulting ligated DNA fragment comprising the five synthetic genes representing Y-MSPe is then subcloned into the pRSET bacterial expression vector (Invitrogen), 3′ to its 6xHis tag, via the NheI and HindIII restriction sites. DNA sequence analysis is performed using the pRSET-specific primers to confirm the Y-MSPe DNA sequence as an open reading frame with the ATG of the pRSET expression vector.
  • the Y-MSPe DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 225 and SEQ ID NO: 226, respectively.
  • the MS-related truncated Y-MSPe gene coding only for preferred epitopes likely to be more frequently recognized in disease on each of the autoantigens selected is prepared as follows: the pGEM-T/shPLP is cleaved at BamHI and BclI and the small BamHI/BclI fragment is removed by gel electrophoresis. The pGEM-T/shPLP is then religated via the compatible BamHI/BclI sites to generate the pGEM-T/sh ⁇ PLP.
  • the XhoI/HindIII DNA fragment comprising the sh ⁇ PLP gene is excised out from the pGEM-T/sh ⁇ PLP and ligated into the pRSET/Y-MSPe digested with XhoI and HindIII to replace the shPLP gene.
  • the resulting plasmid (pRSET/Y-MSPe with shPLP) is then digested with NheI and HindIII and the excised DNA fragment (Y-MSPe with sh ⁇ PLP) is sequentially cleaved and religated via the compatible restriction sites BglII and BamHI, followed by XbaI and SpeI, followed by NsiI and PstI, as depicted in FIG. 7 .
  • the resulting DNA fragment comprising the sh ⁇ symthetic genes is cloned back into the pRSET vector via the NheI and HindIII sites.
  • the resulting plasmid is the pRSET/ ⁇ Y-MSPe ( ⁇ Y-MSPe).
  • DNA sequence analysis is performed using the pRSET-specific primers to confirm the ⁇ Y-MSPe DNA sequence as an open reading frame with the ATG of the pRSET expression vector.
  • the ⁇ Y-MSPe DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 227 and SEQ ID NO: 228, respectively.
  • the Y-MSP-AL and ⁇ Y-MSP-AL genes are constructed in exactly the same manner as described in Examples 10-11 above, according to the schemes depicted in FIGS. 20 and 21 , respectively.
  • the Y-MSP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 92 and SEQ ID NO: 229, respectively.
  • the ⁇ Y-MSP-AL DNA sequence and derived amino acid sequence are represented by SEQ ID NO: 93 and SEQ ID NO: 230, respectively.
  • the invention further provides a synthetic polypeptide comprising amino acid sequences of at least two IECs of a sole autoantigen related to MS or at least one IEC of at least two different autoantigens related to MS and analogs thereof, wherein said amino acid sequences are selected from the sequences of the unaltered and/or altered peptides of the invention.
  • the polypeptide comprises the unaltered amino acid sequences of at least two IECs of a sole autoantigen related to MS consisting of the polypeptides shMOG (SEQ ID NO:103), shMBP (SEQ ID NO:115), shOSP (SEQ ID NO:129), shMOBP (SEQ ID NO:139), and shPLP (SEQ ID NO:155).
  • the polypeptide comprises the unaltered amino acid sequences of at least one IEC of at least two different autoantigens related to MS, preferably the polypeptides Y-MSPe (SEQ ID NO:226) and ⁇ Y-MSPe (SEQ ID NO:228).
  • polypeptides comprise the sequences of the altered peptides of the invention and include, for example:
  • a polypeptide comprising at least two, preferably all the six MOG-AL amino acid sequences of the SEQ ID NOs: 48-53, each of them carrying 1-3 alanine substitutions, preferably containing all the six sequences, more preferably the polypeptide of the amino acid sequence of SEQ ID NO: 166;
  • polypeptide comprising at least two, preferably all the eight MBP-AL amino acid sequences of the SEQ ID NOs: 54-61, each of them carrying 1-3 alanine substitutions, preferably containing all the eight sequences, more preferably the polypeptide of the amino acid sequence of SEQ ID NO: 179;
  • polypeptide comprising at least two, preferably all the eight OSP-AL amino acid sequences of the SEQ ID NOs: 62-69, each of them carrying 1-4 alanine substitutions, preferably containing all the eight sequences, more preferably the polypeptide of the amino acid sequence of SEQ ID NO: 194;
  • a polypeptide comprising at least two, preferably all the four of the MOBP-AL amino acid sequences of the SEQ ID NOs: 70-73, each of them carrying 1-3 alanine substitutions, preferably comprising the four sequences, more preferably the polypeptide of the amino acid sequence of SEQ ID NO: 203;
  • a polypeptide comprising at least two, preferably all the 13 of the PLP-AL amino acid sequences of the SEQ ID NOs: 74-86, each of them carrying 1-3 alanine substitutions, more preferably the polypeptide of the amino acid sequence of SEQ ID NO: 224;
  • a polypeptide comprising: (a) at least two, preferably all the six MOG-AL amino acid sequences of the SEQ ID NOs: 48-53; (b) at least two, preferably all the eight MBP-AL amino acid sequences of the SEQ ID NOs: 54-61; (c) at least two, preferably all the eight OSP-AL amino acid sequences of the SEQ ID NOs: 62-69; (d) at least two, preferably all the four of the MOBP-AL amino acid sequences of the SEQ ID NOs: 70-73; and (e) at least two, preferably all the 13 of the PLP-AL amino acid sequences of the SEQ ID NOs: 74-86, each of the amino acid sequences carrying 1-3 alanine substitutions, preferably comprising all said sequences (a)-(e), more preferably the polypeptide of the amino acid sequence of SEQ ID NO: 229; and
  • the synthetic genes of the present invention can be incorporated into expression vector that may be, for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said synthetic gene and optionally a regulator of the promoter.
  • the recombinant expression vector may then be used to transform or transfect suitable host cells such as bacterial cells, e.g. E. coli cells, or eukaryotic cells such as yeast, insect or preferably, mammalian cells, to provide for expression of a polypeptide of the invention.
  • This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression of the polypeptide.
  • the expressed polypeptide is then recovered by extraction from the host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption, and isolated by protein purification methods known in the art, such as metal chelate chromatography, HPLC, antibody-affinity chromatography etc.
  • the synthetic gene of any one of Examples 1-12 above is cleaved out from the pGEM-T/synthetic gene plasmid and subcloned into the bacterial expression vector pRSET (V351-20; Invitrogen, San Diego, Calif., USA) via suitable restriction sites such as NheI and BglII or NheI and HindIII, 3′ to its 6xIis tag, using standard molecular biology techniques. DNA sequence analysis is performed using the pRSET-specific primers to confirm the synthetic gene DNA sequence as an open reading frame with the ATG of the pRSET expression vector.
  • the pRSET/synthetic gene is then transformed into Escherichia coli host (BL21-DE3), and protein expression is induced by isopropyl ⁇ -D-thio-galactopyranoside (IPTG) (RO392; MBI Fermentas AB). After expression is observed, the expressed protein product is isolated under denaturing conditions (8 M urea) by metal chelate affinity chromatography on Ni 2+ nitriloacetic acid (NTA) agarose (30230; Qiagen Inc., Valencia, Calif., USA) according to the manufacturer's protocol. Fractions containing the isolated protein, as evidenced by SDS-PAGE, are pooled and subjected to reducing conditions with ⁇ -mercaptoethanol. The protein is diluted to 50-100 ⁇ g/ml in 8 M urea and allowed to refold by dialysis against gradually decreasing concentrations of urea (8-0 M). Any aggregated protein is removed by centrifugation.
  • IPTG iso
  • the synthetic gene may also be expressed in a mammalian expression vector as described in WO 01/31037.
  • the synthetic gene DNA is cleaved out from the pGEM-T/synthetic gene plasmid with EcoRI and NotI and subcloned into the mammalian expression vector pCDNA 3.1.
  • the pCDNA 3.1/synthetic gene is transfected into mammalian cells, e.g. NIH3T3 mouse fibroblasts, CHO or any other suitable mammalian cells, and the expressed protein product is isolated and examined by standard molecular biology techniques.
  • the present invention further provides pharmaceutical compositions for multiepitope-directed immunomodulation of MS comprising a pharmaceutically acceptable carrier and a mixture of two or more unaltered and/or altered peptides of the invention, or a synthetic gene of the invention or a polypeptide which is a protein product of a synthetic gene of the invention.
  • the synthetic genes and their protein products may be useful for concomitant immunomodulation of potentially pathogenic autoreactivities against different or multiple epitopes of a single or several target antigens in MS, via DNA vaccination or via tolerogenic administration of the protein product of the synthetic gene.
  • the present invention further relates to a pharmaceutical composition for multiepitope-directed immunomodulation of MS comprising a pharmaceutically acceptable carrier and an agent selected from:
  • a mixture of at least two synthetic peptides each peptide comprising amino acid sequences of at least one immunogenic epitope cluster (IEC) of at least one human autoantigen related to multiple sclerosis (MS), said peptide being selected from an unaltered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N- and C-termini, the resulting IEC being capable of stimulating human T cells;
  • IEC immunogenic epitope cluster
  • MS human autoantigen related to multiple sclerosis
  • a mixture of at least two synthetic peptides each peptide comprising amino acid sequences of at least one immunogenic epitope cluster (IEC) of at least one human autoantigen related to multiple sclerosis (MS), said peptide being selected from an altered synthetic peptide comprising at least one nonameric core sequence which fits into the MS-relevant HLA-DR/DQ molecule and is flanked by 2-5 amino acids at its N- and C-termini, in which sequence one to three T-cell receptor (TCR) contact amino acid residues are substituted by a suitable amino acid such as Ala, the resulting IEC altered in the TCR residue being capable of immunomodulating the potentially pathogenic T-cell response against the epitope without risk of exacerbation;
  • TCR T-cell receptor
  • composition of the invention comprising a mixture of peptides will depend upon the route of administration but typically they can be formulated for topical, parenteral, intramuscular, intravenous, intra-peritoneal, intranasal inhalation, lung inhalation, or intradermal administration.
  • the polypeptide may be administered in an injectable form by mixing with a pharmaceutically acceptable vehicle for an injectable formulation.
  • the pharmaceutically acceptable carrier or diluent may be, for example, sterile isotonic saline solutions, or other isotonic solutions such as phosphate-buffered saline.
  • Additional ingredients of the formulations include suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • the physician will determine the actual dosage, which will be most suitable for an individual patient, and it will vary with the age, weight and response of the particular patient.
  • compositions are administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents.
  • excipients such as starch or lactose
  • capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents.
  • compositions as well as the polypeptides alone can also be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously.
  • the compositions will comprise a suitable carrier or diluent.
  • the compositions are best used in the form of a sterile aqueous solution, which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • the present invention also provides pharmaceutical compositions for the treatment of MS comprising at least one synthetic gene of the invention to be administered via “naked DNA” vaccination or together with a suitable gene delivery vehicle.
  • the gene delivery vehicle may be a non-viral vehicle such as cationic liposomes or a cationic lipid vehicle, or a viral vehicle such as adenovirus vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector or a baculoviral vector.
  • the gene can be, for example, integrated in a retroviral expression vector.
  • the composition of the invention When injected in a soluble tolerogenic route (s.c., i.v. or i.p.) or administered by oral or nasal application, the composition of the invention is expected to downregulate the potentially pathogenic autoimmune responses in MS.
  • the synthetic gene of the invention when administered as a naked DNA constructed into an appropriate malian expression vector, can be effective in vaccinating against the disease.
  • the unaltered and altered peptides as well as the polypeptides comprising sequences of the unaltered and altered peptides of the invention can also be used for diagnosis and/or for monitoring the progression of MS by measuring the levels of immunoactivation of T-cells specific for the autoantigen(s) associated with the disease.
  • T-cell responses to the peptides or polypeptides are likely to be higher in patients than in control individuals, and measurement of such responses by blood cells or serum can be used as a diagnostic/monitoring tool.
  • the levels of T-cell autoreactivity to the peptides or polypeptides are measured by incubating PBLs isolated from peripheral blood in the presence of the relevant peptides or polypeptides, and monitoring the activation of the reactive T-cells by detection of T-cell proliferation, cytokine release and expression of cytokine receptors and other activation-associated cell surface markers.
  • Such assays of T-cell activation are well known to those of skill in the art.
  • MOG APLs Substituted at Crucial TCR Contact Residues 41 and 44 Suppress EAE Induced with MOG 37-52
  • the altered peptides of the invention were tested for their biological activity in relevance to MS, using EAE as a model system.
  • C3H.SW mice Female 2-3 months old C3H.SW mice (Jackson Laboratory, Bar harbor, Maine, USA) were immunized with MOG 37-52 (150 ⁇ g emulsified with CFA supplemented with 300 ⁇ g Mycobacterium tuberculosis ).
  • mice On days 5, 7, 9 and 12 after encephalitogenic challenge, the mice were injected intravenously (i.v.) with an aqueous solution of MOG 37-52 (SEQ ID NO: 231), MOG 37-A44-52 (SEQ ID NO: 232), or MOG 37-Q41-52 (SEQ ID NO: 233), (400 ⁇ g in PBS), or with a combination of MOG 37-A44-52+MOG 37-Q41-52, or with PBS alone. Mice were followed and scored daily for clinical effects on a scale of 0-6. The results in FIG.
  • Y-MSPb the purified protein product of the synthetic Y-MSPb gene, disclosed in WO 01-31037, designed to encode tandemly arranged disease-relevant epitopes clusters of the encephalitogenic proteins MBP, PLP, MOG, and MOBP, was tested for its immunomodulatory effect on EAE associated with multiple pathogenic autoreactivities, upon tolerogenic administration, in the same way as described for Y-MSPa in WO 01-31037.
  • Line T-cells were selected in vitro with Y-MSPb from lymph node cells (LNCs) of (C3H.SW ⁇ SJL/J)F 1 mice immunized with Y-MSPb (containing encephalitogenic epitopes of MOG, MBP, PLP and MOBP) in CFA, and were analyzed after primary response (left panel) and after four cycles of selection (right panel) for their proliferative response to Y-MSPb, phMOG 34-56, phMOG 1-22, shMOG/E, PLP 139-151, shPLP/E, MBP 87-101 and shMBP/E (10 ⁇ /ml).
  • the proliferative response to purified protein derivative (PPD) (5 ⁇ /ml) was analyzed as a measure of specificity. The results are shown in FIG. 9A .
  • the line T cells are directed against each of the myelin autoantigens.
  • FIG. 9B shows that EAE associated with multiple pathogenic autoreactivities induced by Y-MSPb-reactive line T-cells (2 ⁇ 10 ⁇ 6 cells, i.v., on day 0) is fully abrogated by tolerogenic administration of Y-MSPb in PBS given daily intraperitoneally (i.p.) from the day of T-cell transfer.
  • MOG 37-52 or PLP139-151 Targeting the T-cells reacting against only one of the epitopes, MOG 37-52 or PLP139-151, recognized by the multireactive Y-MSPb-elicited line T-cells, has no significant effect on disease incidence or severity, while dual targeting with a combination of MOG 37-52 and PLP139-151 (indicated as MOG+PLP) has only a marginal effect on disease severity.
  • the multitarget autoantigen genes of the invention Y-MSPe, ⁇ Y-MSPe, and preferably the Y-MSP-AL and ⁇ Y-MSP-AL genes can be tested, and their ability to immunomodulate potentially pathogenic autoreactivities against multiple epitopes of several antigens associated with MS, can be shown.

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US9180174B2 (en) 2008-01-25 2015-11-10 Vianex S.A. Conjugates comprising mannan and myelin oligodentrocyte glycoprotein (MOG)
RU2448685C2 (ru) * 2009-11-30 2012-04-27 Российская Федерация в лице Министерства промышленности и торговли Российской Федерации Липосомы, содержащие олигопептиды - фрагменты основного белка миелина, фармацевтическая композиция и способ лечения рассеянного склероза
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